Carboxyl-terminal sequences in APOA5 are important for suppressing ANGPTL3/8 activity.

Apolipoprotein AV (APOA5) lowers plasma triglyceride (TG) levels by binding to the angiopoietin-like protein 3/8 complex (ANGPTL3/8) and suppressing its capacity to inhibit lipoprotein lipase (LPL) catalytic activity and its ability to detach LPL from binding sites within capillaries. However, the sequences in APOA5 that are required for suppressing ANGPTL3/8 activity have never been defined. A clue to the identity of those sequences was the presence of severe hypertriglyceridemia in two patients harboring an APOA5 mutation that truncates APOA5 by 35 residues ("APOA5Δ35"). We found that wild-type (WT) human APOA5, but not APOA5Δ35, suppressed ANGPTL3/8's ability to inhibit LPL catalytic activity. To pursue that finding, we prepared a mutant mouse APOA5 protein lacking 40 C-terminal amino acids ("APOA5Δ40"). Mouse WT-APOA5, but not APOA5Δ40, suppressed ANGPTL3/8's capacity to inhibit LPL catalytic activity and sharply reduced plasma TG levels in mice. WT-APOA5, but not APOA5Δ40, increased intracapillary LPL levels and reduced plasma TG levels in Apoa5-/- mice (where TG levels are high and intravascular LPL levels are low). Also, WT-APOA5, but not APOA5Δ40, blocked the ability of ANGPTL3/8 to detach LPL from cultured cells. Finally, an antibody against a synthetic peptide corresponding to the last 26 amino acids of mouse APOA5 reduced intracapillary LPL levels and increased plasma TG levels in WT mice. We conclude that C-terminal sequences in APOA5 are crucial for suppressing ANGPTL3/8 activity in vitro and for regulating intracapillary LPL levels and plasma TG levels in vivo.

Inverse effects of APOC2 and ANGPTL4 on the conformational dynamics of lid-anchoring structures in lipoprotein lipase.

The lipolytic processing of triglyceride-rich lipoproteins (TRLs) by lipoprotein lipase (LPL) is crucial for the delivery of dietary lipids to the heart, skeletal muscle, and adipose tissue. The processing of TRLs by LPL is regulated in a tissue-specific manner by a complex interplay between activators and inhibitors. Angiopoietin-like protein 4 (ANGPTL4) inhibits LPL by reducing its thermal stability and catalyzing the irreversible unfolding of LPL's α/ß-hydrolase domain. We previously mapped the ANGPTL4 binding site on LPL and defined the downstream unfolding events resulting in LPL inactivation. The binding of LPL to glycosylphosphatidylinositol-anchored high-density lipoprotein-binding protein 1 protects against LPL unfolding. The binding site on LPL for an activating cofactor, apolipoprotein C2 (APOC2), and the mechanisms by which APOC2 activates LPL have been unclear and controversial. Using hydrogen-deuterium exchange/mass spectrometry, we now show that APOC2's C-terminal α-helix binds to regions of LPL surrounding the catalytic pocket. Remarkably, APOC2's binding site on LPL overlaps with that for ANGPTL4, but their effects on LPL conformation are distinct. In contrast to ANGPTL4, APOC2 increases the thermal stability of LPL and protects it from unfolding. Also, the regions of LPL that anchor the lid are stabilized by APOC2 but destabilized by ANGPTL4, providing a plausible explanation for why APOC2 is an activator of LPL, while ANGPTL4 is an inhibitor. Our studies provide fresh insights into the molecular mechanisms by which APOC2 binds and stabilizes LPL-and properties that we suspect are relevant to the conformational gating of LPL's active site.

Intracapillary LPL levels in brown adipose tissue, visualized with an antibody-based approach, are regulated by ANGPTL4 at thermoneutral temperatures.

Lipoprotein lipase (LPL) is secreted into the interstitial spaces by parenchymal cells and then transported into capillaries by GPIHBP1. LPL carries out the lipolytic processing of triglyceride (TG)-rich lipoproteins (TRLs), but the tissue-specific regulation of LPL is incompletely understood. Plasma levels of TG hydrolase activity after heparin injection are often used to draw inferences about intravascular LPL levels, but the validity of these inferences is unclear. Moreover, plasma TG hydrolase activity levels are not helpful for understanding LPL regulation in specific tissues. Here, we sought to elucidate LPL regulation under thermoneutral conditions (30 °C). To pursue this objective, we developed an antibody-based method to quantify (in a direct fashion) LPL levels inside capillaries. At 30 °C, intracapillary LPL levels fell sharply in brown adipose tissue (BAT) but not heart. The reduced intracapillary LPL levels were accompanied by reduced margination of TRLs along capillaries. ANGPTL4 expression in BAT increased fourfold at 30 °C, suggesting a potential explanation for the lower intracapillary LPL levels. Consistent with that idea, Angptl4 deficiency normalized both LPL levels and TRL margination in BAT at 30 °C. In Gpihbp1-/- mice housed at 30 °C, we observed an ANGPTL4-dependent decrease in LPL levels within the interstitial spaces of BAT, providing in vivo proof that ANGPTL4 regulates LPL levels before LPL transport into capillaries. In conclusion, our studies have illuminated intracapillary LPL regulation under thermoneutral conditions. Our approaches will be useful for defining the impact of genetic variation and metabolic disease on intracapillary LPL levels and TRL processing.

The lipoprotein lipase that is shuttled into capillaries by GPIHBP1 enters the glycocalyx where it mediates lipoprotein processing.

Lipoprotein lipase (LPL), the enzyme that carries out the lipolytic processing of triglyceride-rich lipoproteins (TRLs), is synthesized by adipocytes and myocytes and secreted into the interstitial spaces. The LPL is then bound by GPIHBP1, a GPI-anchored protein of endothelial cells (ECs), and transported across ECs to the capillary lumen. The assumption has been that the LPL that is moved into capillaries remains attached to GPIHBP1 and that GPIHBP1 serves as a platform for TRL processing. In the current studies, we examined the validity of that assumption. We found that an LPL-specific monoclonal antibody (mAb), 88B8, which lacks the ability to detect GPIHBP1-bound LPL, binds avidly to LPL within capillaries. We further demonstrated, by confocal microscopy, immunogold electron microscopy, and nanoscale secondary ion mass spectrometry analyses, that the LPL detected by mAb 88B8 is located within the EC glycocalyx, distant from the GPIHBP1 on the EC plasma membrane. The LPL within the glycocalyx mediates the margination of TRLs along capillaries and is active in TRL processing, resulting in the delivery of lipoprotein-derived lipids to immediately adjacent parenchymal cells. Thus, the LPL that GPIHBP1 transports into capillaries can detach and move into the EC glycocalyx, where it functions in the intravascular processing of TRLs.

CL-11 circulates in serum as functionally distinct isoforms.

Collectin-11 (CL-11) is a pattern recognition molecule of the lectin pathway capable of interacting with collectin-10 (CL-10) and the MASPs to activate the complement cascade. Alternative splicing of the COLEC11 gene gives rise to two different isoforms found in serum (A and D). These isoforms vary in the length of their collagen-like region, which is involved in the stabilization of the trimeric subunit and the interaction with the MASPs. Here we aim at elucidating the biological differences of naturally occurring CL-11 isoforms A and D. We produced recombinant CL-11 as independent isoforms (CL-11A and CL-11D) and together with CL-10 (CL-10/11A, CL-10/11D). Both CL-11 isoforms associated with CL-10, but CL-11D did so to a lesser extent. CL-10/11 heterocomplexes were composed of trimeric subunits of CL-10 and CL-11, as opposed to CL-10 and CL-11 homotrimers. Heterocomplexes were more stable and migrated with higher apparent molecular weights. Immunoprecipitation of serum CL-11 and subsequent mass spectrometry analysis confirmed that native CL-11 circulates in the form of CL-10/11 heterocomplexes that associate with MASP-1, and MASP-3, but not necessarily MASP-2. Despite a shorter collagen region, CL-11D was capable to bind to the MASPs, suggesting that the missing exon 4 is not required for MASP association CL-11D had a reduced ligand binding compared to full-length CL-11A. Based on its reduced ability to oligomerize, form CL-10/11 heterocomplexes, and bind to ligands, we hypothesize that CL-11D may have a limited complement activation potential compared to full-length CL-11A.

A protein of capillary endothelial cells, GPIHBP1, is crucial for plasma triglyceride metabolism.

GPIHBP1, a protein of capillary endothelial cells (ECs), is a crucial partner for lipoprotein lipase (LPL) in the lipolytic processing of triglyceride-rich lipoproteins. GPIHBP1, which contains a three-fingered cysteine-rich LU (Ly6/uPAR) domain and an intrinsically disordered acidic domain (AD), captures LPL from within the interstitial spaces (where it is secreted by parenchymal cells) and shuttles it across ECs to the capillary lumen. Without GPIHBP1, LPL remains stranded within the interstitial spaces, causing severe hypertriglyceridemia (chylomicronemia). Biophysical studies revealed that GPIHBP1 stabilizes LPL structure and preserves LPL activity. That discovery was the key to crystallizing the GPIHBP1-LPL complex. The crystal structure revealed that GPIHBP1's LU domain binds, largely by hydrophobic contacts, to LPL's C-terminal lipid-binding domain and that the AD is positioned to project across and interact, by electrostatic forces, with a large basic patch spanning LPL's lipid-binding and catalytic domains. We uncovered three functions for GPIHBP1's AD. First, it accelerates the kinetics of LPL binding. Second, it preserves LPL activity by inhibiting unfolding of LPL's catalytic domain. Third, by sheathing LPL's basic patch, the AD makes it possible for LPL to move across ECs to the capillary lumen. Without the AD, GPIHBP1-bound LPL is trapped by persistent interactions between LPL and negatively charged heparan sulfate proteoglycans (HSPGs) on the abluminal surface of ECs. The AD interrupts the HSPG interactions, freeing LPL-GPIHBP1 complexes to move across ECs to the capillary lumen. GPIHBP1 is medically important; GPIHBP1 mutations cause lifelong chylomicronemia, and GPIHBP1 autoantibodies cause some acquired cases of chylomicronemia.

APOA5 Deficiency Causes Hypertriglyceridemia by Reducing Amounts of Lipoprotein Lipase in Capillaries.

Apolipoprotein AV (APOA5) deficiency causes hypertriglyceridemia in mice and humans. For years, the cause remained a mystery, but the mechanisms have now come into focus. Here, we review progress in defining APOA5's function in plasma triglyceride metabolism. Biochemical studies revealed that APOA5 binds to the angiopoietin-like protein 3/8 complex (ANGPTL3/8) and suppresses its ability to inhibit the activity of lipoprotein lipase (LPL). Thus, APOA5 deficiency is accompanied by increased ANGPTL3/8 activity and lower levels of LPL activity. APOA5 deficiency also reduces amounts of LPL in capillaries of oxidative tissues (e.g., heart, brown adipose tissue). Cell culture experiments revealed the likely explanation: ANGPTL3/8 detaches LPL from its binding sites on the surface of cells, and that effect is blocked by APOA5. Both the low intracapillary LPL levels and the high plasma triglyceride levels in Apoa5-/- mice are normalized by recombinant APOA5. Carboxyl-terminal sequences in APOA5 are crucial for its function; a mutant APOA5 lacking 40-carboxyl-terminal residues cannot bind to ANGPTL3/8 and lacks the ability to change intracapillary LPL levels or plasma triglyceride levels in Apoa5-/- mice. Also, an antibody against the last 26 amino acids of APOA5 reduces intracapillary LPL levels and increases plasma triglyceride levels in wild-type mice. An inhibitory ANGPTL3/8-specific antibody functions as an APOA5-mimetic reagent, increasing intracapillary LPL levels and lowering plasma triglyceride levels in both Apoa5-/- and wild-type mice. That antibody is a potentially attractive strategy for treating elevated plasma lipid levels in human patients.

Quantification of lipoprotein lipase in mouse plasma with a sandwich enzyme-linked immunosorbent assay.

To support in vivo and in vitro studies of intravascular triglyceride metabolism in mice, we created rat monoclonal antibodies (mAbs) against mouse LPL. Two mAbs, mAbs 23A1 and 31A5, were used to develop a sandwich ELISA for mouse LPL. The detection of mouse LPL by the ELISA was linear in concentrations ranging from 0.31 ng/ml to 20 ng/ml. The sensitivity of the ELISA made it possible to quantify LPL in serum and in both pre-heparin and post-heparin plasma samples (including in grossly lipemic samples). LPL mass and activity levels in the post-heparin plasma were lower in Gpihbp1-/- mice than in wild-type mice. In both groups of mice, LPL mass and activity levels were positively correlated. Our mAb-based sandwich ELISA for mouse LPL will be useful for any investigator who uses mouse models to study LPL-mediated intravascular lipolysis.

The intrinsic instability of the hydrolase domain of lipoprotein lipase facilitates its inactivation by ANGPTL4-catalyzed unfolding.

The complex between lipoprotein lipase (LPL) and its endothelial receptor (GPIHBP1) is responsible for the lipolytic processing of triglyceride-rich lipoproteins (TRLs) along the capillary lumen, a physiologic process that releases lipid nutrients for vital organs such as heart and skeletal muscle. LPL activity is regulated in a tissue-specific manner by endogenous inhibitors (angiopoietin-like [ANGPTL] proteins 3, 4, and 8), but the molecular mechanisms are incompletely understood. ANGPTL4 catalyzes the inactivation of LPL monomers by triggering the irreversible unfolding of LPL's α/ß-hydrolase domain. Here, we show that this unfolding is initiated by the binding of ANGPTL4 to sequences near LPL's catalytic site, including ß2, ß3-α3, and the lid. Using pulse-labeling hydrogen‒deuterium exchange mass spectrometry, we found that ANGPTL4 binding initiates conformational changes that are nucleated on ß3-α3 and progress to ß5 and ß4-α4, ultimately leading to the irreversible unfolding of regions that form LPL's catalytic pocket. LPL unfolding is context dependent and varies with the thermal stability of LPL's α/ß-hydrolase domain (Tm of 34.8 °C). GPIHBP1 binding dramatically increases LPL stability (Tm of 57.6 °C), while ANGPTL4 lowers the onset of LPL unfolding by ∼20 °C, both for LPL and LPL•GPIHBP1 complexes. These observations explain why the binding of GPIHBP1 to LPL retards the kinetics of ANGPTL4-mediated LPL inactivation at 37 °C but does not fully suppress inactivation. The allosteric mechanism by which ANGPTL4 catalyzes the irreversible unfolding and inactivation of LPL is an unprecedented pathway for regulating intravascular lipid metabolism.

Unfolding of monomeric lipoprotein lipase by ANGPTL4: Insight into the regulation of plasma triglyceride metabolism.

The binding of lipoprotein lipase (LPL) to GPIHBP1 focuses the intravascular hydrolysis of triglyceride-rich lipoproteins on the surface of capillary endothelial cells. This process provides essential lipid nutrients for vital tissues (e.g., heart, skeletal muscle, and adipose tissue). Deficiencies in either LPL or GPIHBP1 impair triglyceride hydrolysis, resulting in severe hypertriglyceridemia. The activity of LPL in tissues is regulated by angiopoietin-like proteins 3, 4, and 8 (ANGPTL). Dogma has held that these ANGPTLs inactivate LPL by converting LPL homodimers into monomers, rendering them highly susceptible to spontaneous unfolding and loss of enzymatic activity. Here, we show that binding of an LPL-specific monoclonal antibody (5D2) to the tryptophan-rich lipid-binding loop in the carboxyl terminus of LPL prevents homodimer formation and forces LPL into a monomeric state. Of note, 5D2-bound LPL monomers are as stable as LPL homodimers (i.e., they are not more prone to unfolding), but they remain highly susceptible to ANGPTL4-catalyzed unfolding and inactivation. Binding of GPIHBP1 to LPL alone or to 5D2-bound LPL counteracts ANGPTL4-mediated unfolding of LPL. In conclusion, ANGPTL4-mediated inactivation of LPL, accomplished by catalyzing the unfolding of LPL, does not require the conversion of LPL homodimers into monomers. Thus, our findings necessitate changes to long-standing dogma on mechanisms for LPL inactivation by ANGPTL proteins. At the same time, our findings align well with insights into LPL function from the recent crystal structure of the LPL•GPIHBP1 complex.

ANGPTL4: a new mode in the regulation of intravascular lipolysis.

PURPOSE OF REVIEW: Lipoprotein lipase (LPL) is the rate-limiting enzyme for intravascular processing of circulating triglyceride-rich lipoproteins (TRLs). One emerging strategy for therapeutic lowering of plasma triglyceride levels aims at increasing the longevity of LPL activity by attenuating its inhibition from angiopoietin-like proteins (ANGPTL) 3, 4 and 8. This mini-review focuses on recent insights into the molecular mechanisms underpinning the regulation of LPL activity in the intravascular unit by ANGPTLs with special emphasis on ANGPTL4. RECENT FINDINGS: Our knowledge on the molecular interplays between LPL, its endothelial transporter GPIHBP1, and its inhibitor(s) ANGPTL4, ANGPTL3 and ANGPTL8 have advanced considerably in the last 2 years and provides an outlined on how these proteins regulate the activity and compartmentalization of LPL. A decisive determinant instigating this control is the inherent protein instability of LPL at normal body temperature, a property that is reciprocally impacted by the binding of GPIHBP1 and ANGPTLs. Additional layers in this complex LPL regulation is provided by the different modulation of ANGPTL4 and ANGPTL3 activities by ANGPTL8 and the inhibition of ANGPTL3/8 complexes by apolipoprotein A5 (APOA5). SUMMARY: Posttranslational regulation of LPL activity in the intravascular space is essential for the differential partitioning of TRLs across tissues and their lipolytic processing in response to nutritional cues.

Disorder in a two-domain neuronal Ca2+-binding protein regulates domain stability and dynamics using ligand mimicry.

Understanding the interplay between sequence, structure and function of proteins has been complicated in recent years by the discovery of intrinsically disordered proteins (IDPs), which perform biological functions in the absence of a well-defined three-dimensional fold. Disordered protein sequences account for roughly 30% of the human proteome and in many proteins, disordered and ordered domains coexist. However, few studies have assessed how either feature affects the properties of the other. In this study, we examine the role of a disordered tail in the overall properties of the two-domain, calcium-sensing protein neuronal calcium sensor 1 (NCS-1). We show that loss of just six of the 190 residues at the flexible C-terminus is sufficient to severely affect stability, dynamics, and folding behavior of both ordered domains. We identify specific hydrophobic contacts mediated by the disordered tail that may be responsible for stabilizing the distal N-terminal domain. Moreover, sequence analyses indicate the presence of an LSL-motif in the tail that acts as a mimic of native ligands critical to the observed order-disorder communication. Removing the disordered tail leads to a shorter life-time of the ligand-bound complex likely originating from the observed destabilization. This close relationship between order and disorder may have important implications for how investigations into mixed systems are designed and opens up a novel avenue of drug targeting exploiting this type of behavior.

Structure of the lipoprotein lipase-GPIHBP1 complex that mediates plasma triglyceride hydrolysis.

Lipoprotein lipase (LPL) is responsible for the intravascular processing of triglyceride-rich lipoproteins. The LPL within capillaries is bound to GPIHBP1, an endothelial cell protein with a three-fingered LU domain and an N-terminal intrinsically disordered acidic domain. Loss-of-function mutations in LPL or GPIHBP1 cause severe hypertriglyceridemia (chylomicronemia), but structures for LPL and GPIHBP1 have remained elusive. Inspired by our recent discovery that GPIHBP1's acidic domain preserves LPL structure and activity, we crystallized an LPL-GPIHBP1 complex and solved its structure. GPIHBP1's LU domain binds to LPL's C-terminal domain, largely by hydrophobic interactions. Analysis of electrostatic surfaces revealed that LPL contains a large basic patch spanning its N- and C-terminal domains. GPIHBP1's acidic domain was not defined in the electron density map but was positioned to interact with LPL's large basic patch, providing a likely explanation for how GPIHBP1 stabilizes LPL. The LPL-GPIHBP1 structure provides insights into mutations causing chylomicronemia.

Lipoprotein lipase is active as a monomer.

Lipoprotein lipase (LPL), the enzyme that hydrolyzes triglycerides in plasma lipoproteins, is assumed to be active only as a homodimer. In support of this idea, several groups have reported that the size of LPL, as measured by density gradient ultracentrifugation, is ∼110 kDa, twice the size of LPL monomers (∼55 kDa). Of note, however, in those studies the LPL had been incubated with heparin, a polyanionic substance that binds and stabilizes LPL. Here we revisited the assumption that LPL is active only as a homodimer. When freshly secreted human LPL (or purified preparations of LPL) was subjected to density gradient ultracentrifugation (in the absence of heparin), LPL mass and activity peaks exhibited the size expected of monomers (near the 66-kDa albumin standard). GPIHBP1-bound LPL also exhibited the size expected for a monomer. In the presence of heparin, LPL size increased, overlapping with a 97.2-kDa standard. We also used density gradient ultracentrifugation to characterize the LPL within the high-salt and low-salt peaks from a heparin-Sepharose column. The catalytically active LPL within the high-salt peak exhibited the size of monomers, whereas most of the inactive LPL in the low-salt peak was at the bottom of the tube (in aggregates). Consistent with those findings, the LPL in the low-salt peak, but not that in the high-salt peak, was easily detectable with single mAb sandwich ELISAs, in which LPL is captured and detected with the same antibody. We conclude that catalytically active LPL can exist in a monomeric state.

Expression and one-step purification of active LPL contemplated by biophysical considerations.

LPL is essential for intravascular lipid metabolism and is of high medical relevance. Since LPL is notoriously unstable, there is an unmet need for a robust expression system producing high quantities of active and pure recombinant human LPL (hLPL). We showed previously that bovine LPL purified from milk is unstable at body temperature (Tm is 34.8°C), but in the presence of the endothelial transporter glycosylphosphatidylinositol-anchored high density lipoprotein-binding protein 1 (GPIHBP1), LPL is stabile (Tm increases to 57.6°C). Building on this information, we now designed an expression system for hLPL using Drosophila Schneider 2 cells grown in suspension at high cell density and at an advantageous temperature of 25°C. We cotransfected Schneider 2 cells with hLPL, lipase maturation factor 1, and soluble GPIHBP1 to provide an efficient chaperoning and stabilization of LPL in all compartments during synthesis and after secretion into the conditioned medium. For LPL purification, we used heparin-Sepharose affinity chromatography, which disrupted LPL-GPIHBP1 complexes causing GPIHBP1 to elute with the flow-through of the conditioned media. This one-step purification procedure yielded high quantities of pure and active LPL (4-28 mg/l). Purification of several hLPL variants (furin cleavage-resistant mutant R297A, active-site mutant S132A, and lipid-binding-deficient mutant W390A-W393A-W394A) as well as murine LPL underscores the versatility and robustness of this protocol. Notably, we were able to produce and purify LPL containing the cognate furin cleavage site. This method provides an efficient and cost-effective approach to produce large quantities of LPL for biophysical and large-scale drug discovery studies.

A disordered acidic domain in GPIHBP1 harboring a sulfated tyrosine regulates lipoprotein lipase.

The intravascular processing of triglyceride-rich lipoproteins depends on lipoprotein lipase (LPL) and GPIHBP1, a membrane protein of endothelial cells that binds LPL within the subendothelial spaces and shuttles it to the capillary lumen. In the absence of GPIHBP1, LPL remains mislocalized within the subendothelial spaces, causing severe hypertriglyceridemia (chylomicronemia). The N-terminal domain of GPIHBP1, an intrinsically disordered region (IDR) rich in acidic residues, is important for stabilizing LPL's catalytic domain against spontaneous and ANGPTL4-catalyzed unfolding. Here, we define several important properties of GPIHBP1's IDR. First, a conserved tyrosine in the middle of the IDR is posttranslationally modified by O-sulfation; this modification increases both the affinity of GPIHBP1-LPL interactions and the ability of GPIHBP1 to protect LPL against ANGPTL4-catalyzed unfolding. Second, the acidic IDR of GPIHBP1 increases the probability of a GPIHBP1-LPL encounter via electrostatic steering, increasing the association rate constant (kon) for LPL binding by >250-fold. Third, we show that LPL accumulates near capillary endothelial cells even in the absence of GPIHBP1. In wild-type mice, we expect that the accumulation of LPL in close proximity to capillaries would increase interactions with GPIHBP1. Fourth, we found that GPIHBP1's IDR is not a key factor in the pathogenicity of chylomicronemia in patients with the GPIHBP1 autoimmune syndrome. Finally, based on biophysical studies, we propose that the negatively charged IDR of GPIHBP1 traverses a vast space, facilitating capture of LPL by capillary endothelial cells and simultaneously contributing to GPIHBP1's ability to preserve LPL structure and activity.

Chylomicronemia from GPIHBP1 autoantibodies.

Some cases of chylomicronemia are caused by autoantibodies against glycosylphosphatidylinositol-anchored HDL binding protein 1 (GPIHBP1), an endothelial cell protein that shuttles LPL to the capillary lumen. GPIHBP1 autoantibodies prevent binding and transport of LPL by GPIHBP1, thereby disrupting the lipolytic processing of triglyceride-rich lipoproteins. Here, we review the "GPIHBP1 autoantibody syndrome" and summarize clinical and laboratory findings in 22 patients. All patients had GPIHBP1 autoantibodies and chylomicronemia, but we did not find a correlation between triglyceride levels and autoantibody levels. Many of the patients had a history of pancreatitis, and most had clinical and/or serological evidence of autoimmune disease. IgA autoantibodies were present in all patients, and IgG4 autoantibodies were present in 19 of 22 patients. Patients with GPIHBP1 autoantibodies had low plasma LPL levels, consistent with impaired delivery of LPL into capillaries. Plasma levels of GPIHBP1, measured with a monoclonal antibody-based ELISA, were very low in 17 patients, reflecting the inability of the ELISA to detect GPIHBP1 in the presence of autoantibodies (immunoassay interference). However, GPIHBP1 levels were very high in five patients, indicating little capacity of their autoantibodies to interfere with the ELISA. Recently, several GPIHBP1 autoantibody syndrome patients were treated successfully with rituximab, resulting in the disappearance of GPIHBP1 autoantibodies and normalization of both plasma triglyceride and LPL levels. The GPIHBP1 autoantibody syndrome should be considered in any patient with newly acquired and unexplained chylomicronemia.

The structural basis for monoclonal antibody 5D2 binding to the tryptophan-rich loop of lipoprotein lipase.

For three decades, the LPL-specific monoclonal antibody 5D2 has been used to investigate LPL structure/function and intravascular lipolysis. 5D2 has been used to measure LPL levels, block the triglyceride hydrolase activity of LPL, and prevent the propensity of concentrated LPL preparations to form homodimers. Two early studies on the location of the 5D2 epitope reached conflicting conclusions, but the more convincing report suggested that 5D2 binds to a tryptophan (Trp)-rich loop in the carboxyl terminus of LPL. The same loop had been implicated in lipoprotein binding. Using surface plasmon resonance, we showed that 5D2 binds with high affinity to a synthetic LPL peptide containing the Trp-rich loop of human (but not mouse) LPL. We also showed, by both fluorescence and UV resonance Raman spectroscopy, that the Trp-rich loop binds lipids. Finally, we used X-ray crystallography to solve the structure of the Trp-rich peptide bound to a 5D2 Fab fragment. The Trp-rich peptide contains a short α-helix, with two Trps projecting into the antigen recognition site. A proline substitution in the α-helix, found in mouse LPL, is expected to interfere with several hydrogen bonds, explaining why 5D2 cannot bind to mouse LPL.

Did evolution create a flexible ligand-binding cavity in the urokinase receptor through deletion of a plesiotypic disulfide bond?

The urokinase receptor (uPAR) is a founding member of a small protein family with multiple Ly6/uPAR (LU) domains. The motif defining these LU domains contains five plesiotypic disulfide bonds stabilizing its prototypical three-fingered fold having three protruding loops. Notwithstanding the detailed knowledge on structure-function relationships in uPAR, one puzzling enigma remains unexplored. Why does the first LU domain in uPAR (DI) lack one of its consensus disulfide bonds, when the absence of this particular disulfide bond impairs the correct folding of other single LU domain-containing proteins? Here, using a variety of contemporary biophysical methods, we found that reintroducing the two missing half-cystines in uPAR DI caused the spontaneous formation of the corresponding consensus 7-8 LU domain disulfide bond. Importantly, constraints due to this cross-link impaired (i) the binding of uPAR to its primary ligand urokinase and (ii) the flexible interdomain assembly of the three LU domains in uPAR. We conclude that the evolutionary deletion of this particular disulfide bond in uPAR DI may have enabled the assembly of a high-affinity urokinase-binding cavity involving all three LU domains in uPAR. Of note, an analogous neofunctionalization occurred in snake venom α-neurotoxins upon loss of another pair of the plesiotypic LU domain half-cystines. In summary, elimination of the 7-8 consensus disulfide bond in the first LU domain of uPAR did have significant functional and structural consequences.

Autoantibodies against GPIHBP1 as a Cause of Hypertriglyceridemia.

BACKGROUND: A protein that is expressed on capillary endothelial cells, called GPIHBP1 (glycosylphosphatidylinositol-anchored high-density lipoprotein binding protein 1), binds lipoprotein lipase and shuttles it to its site of action in the capillary lumen. A deficiency in GPIHBP1 prevents lipoprotein lipase from reaching the capillary lumen. Patients with GPIHBP1 deficiency have low plasma levels of lipoprotein lipase, impaired intravascular hydrolysis of triglycerides, and severe hypertriglyceridemia (chylomicronemia). During the characterization of a monoclonal antibody-based immunoassay for GPIHBP1, we encountered two plasma samples (both from patients with chylomicronemia) that contained an interfering substance that made it impossible to measure GPIHBP1. That finding raised the possibility that those samples might contain GPIHBP1 autoantibodies. METHODS: Using a combination of immunoassays, Western blot analyses, and immunocytochemical studies, we tested the two plasma samples (as well as samples from other patients with chylomicronemia) for the presence of GPIHBP1 autoantibodies. We also tested the ability of GPIHBP1 autoantibodies to block the binding of lipoprotein lipase to GPIHBP1. RESULTS: We identified GPIHBP1 autoantibodies in six patients with chylomicronemia and found that these autoantibodies blocked the binding of lipoprotein lipase to GPIHBP1. As in patients with GPIHBP1 deficiency, those with GPIHBP1 autoantibodies had low plasma levels of lipoprotein lipase. Three of the six patients had systemic lupus erythematosus. One of these patients who had GPIHBP1 autoantibodies delivered a baby with plasma containing maternal GPIHBP1 autoantibodies; the infant had severe but transient chylomicronemia. Two of the patients with chylomicronemia and GPIHBP1 autoantibodies had a response to treatment with immunosuppressive agents. CONCLUSIONS: In six patients with chylomicronemia, GPIHBP1 autoantibodies blocked the ability of GPIHBP1 to bind and transport lipoprotein lipase, thereby interfering with lipoprotein lipase-mediated processing of triglyceride-rich lipoproteins and causing severe hypertriglyceridemia. (Funded by the National Heart, Lung, and Blood Institute and the Leducq Foundation.).